Methods for electrochemical analysis using matrix compositions with alkylphenazine quaternary salt and a nitrosoaniline

ABSTRACT

A chemistry matrix for use in determining the concentration of an analyte in a biological fluid includes a glucose dehydrogenase, nicotinamide adenine dinucleotide, an alkylphenazine quaternary salt, and/or a nitrosoaniline. The chemistry matrix is used with an electrochemical biosensor to determine the concentration of an analyte after a reaction occurs within the biosensor, at which time an analysis is completed to determine the concentration. A method of determining the concentration of an analyte using the chemistry matrix of glucose dehydrogenase, nicotinamide adenine dinucleotide, an alkylphenazine quaternary salt, and/or a nitrosoaniline is another aspect that is described. The method also further features test times of five seconds or less. Methods utilizing the new chemistry matrix can readily determine an analyte such as blood glucose at concentrations of from about 20-600 mg/dL at a pH of from about 6.5 to about 8.5.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/056,473 filed Mar. 27, 2008, now U.S. Pat. No. 8,008,037, which ishereby incorporated by reference.

BACKGROUND Technical Field

The present disclosure generally relates to a chemistry matrix andmethods for measuring the presence and/or concentration of an analyte ina biological fluid. More specifically, but not exclusively, the presentdisclosure relates to a chemistry matrix and methods that increase theanalyte specificity when measuring analyte with an electrochemicalbiosensor in the presence of interfering substances. As used herein, theterm “chemistry matrix” refers to a physical region containing at leastone chemical substance capable of reacting with an analyte.

DESCRIPTION OF RELATED ART

Measuring the concentration of substances, particularly in the presenceof other confounding substances and under varied conditions, isimportant in many fields. For example, the measurement of glucose inbodily fluids, such as blood, under varied conditions and in thepresence of interfering substances, is crucial to the effectivetreatment of diabetes. The failure to properly control blood glucoselevels can produce extreme complications, including blindness and lossof circulation in the extremities, which can ultimately deprive thediabetic of use of his or her fingers, hands, feet, etc.

Test strips are often used to measure the presence and/or concentrationof selected analytes in test samples. For example, a variety of teststrips are used to measure glucose concentrations in blood to monitorthe blood sugar level of people with diabetes. These test strips includea reaction chamber into which a reagent composition has been deposited.Current trends in test strips require smaller test samples and fastertest analysis times. A significant benefit is provided to the patientwhen using smaller test samples, which can be obtained from lesssensitive areas of the body, such as the forearm. Additionally, fasterand more accurate test times provide added convenience and bettercontrol of the patient's blood sugar level.

Several methods are known for measuring the concentration of analytes,such as, for example, glucose, in a blood sample. Such methods typicallyfall into one of two categories: optical methods and electrochemicalmethods. Optical methods generally involve reflectance or absorbancespectroscopy to observe the spectrum shift in a reagent. Such shifts arecaused by a chemical reaction that produces a color change indicative ofthe concentration of the analyte. Electrochemical methods generallyinvolve amperometric, coulometric, potentiometric, and/or conductiveresponses indicative of the concentration of the analyte. See, forexample, U.S. Pat. No. 4,233,029 to Columbus; U.S. Pat. No. 4,225,410 toPace; U.S. Pat. No. 4,323,536 to Columbus; U.S. Pat. No. 4,008,448 toMuggli; 4,654,197 to Lilja et al.; U.S. Pat. No. 5,108,564 to Szuminskyet al.; U.S. Pat. No. 5,120,420 to Nankai et al.; U.S. Pat. No.5,128,015 to Szuminsky et al.; U.S. Pat. No. 5,243,516 to White; U.S.Pat. No. 5,437,999 to Diebold et al.; U.S. Pat. No. 5,288,636 toPollmann et al.; U.S. Pat. No. 5,628,890 to Carter et al.; U.S. Pat. No.5,682,884 to Hill et al.; U.S. Pat. No. 5,727,548 to Hill et al.;5,997,817 to Crismore et al.; U.S. Pat. No. 6,004,441 to Fujiwara etal.; U.S. Pat. No. 4,919,770 to Priedel et al.; and U.S. Pat. No.6,054,039 to Shieh, which are hereby incorporated by reference in theirentireties. Electrochemical methods typically use blood glucose meters(but not always) to measure the electrochemical response of a bloodsample in the presence of a reagent. The reagent reacts with the glucoseto produce charge carriers that are not otherwise present in blood.Consequently, the electrochemical response of the blood in the presenceof a given signal is intended to be primarily dependent upon theconcentration of blood glucose. Typical reagents used in electrochemicalblood glucose meters are disclosed in U.S. Pat. Nos. 5,997,817,5,122,244, and 5,286,362, which are hereby incorporated by reference intheir entireties.

A number of error sources can create inaccurate results when measuringanalyte levels in body fluid. Sometimes harsh conditions to which thesensor is exposed worsen the accuracy of the sensor. Occasionally, thesensor can experience harmful conditions, often termed “strip rotting”or “vial abuse”, which refers to when the sensors during storage areabused and exposed to detrimental conditions, such as excessive heat,light, and/or moisture. This exposure to excessive heat and/or moisturecan result in slowing of the reaction times due to loss of enzymeactivity.

In the past, these issues have been avoided by using enzymes that havevery fast reaction times and high specific activities with the analytebeing measured. By utilizing enzymes with these particularcharacteristics, reactions having high and similar levels of completionunder all test conditions, such as at various temperatures andhematocrit levels, can be achieved. However, as a practical matter,enzymes usually cannot be incorporated with high enough amounts into thesensor without causing a significant loss in performance of the sensor.In addition, enzymes of high specific activities are not alwaysdesirable for all analytes. For example, such systems for determiningglucose levels still fail to address the need of being free from theeffects of interferents, such as maltose, galactose, xylose, and thelike, which can create inaccurate readings.

For example, patients undergoing peritoneal dialysis or IGG therapy canexperience high levels of maltose in their blood, which can interferewith accurate blood glucose readings. Therefore, interference frommaltose can be a significant problem. As an illustration, AbbottLaboratories' Freeystyle® blood glucose monitoring system employs aglucose-dye-oxidoreductase (GlucDOR) enzyme in conjunction with acoulometric technique with a variable test time. However, such a systemis still clinically unacceptable due to interference from maltose, and,as a practical matter, the use of coulometry, as currently practiced,has a number of significant drawbacks. Further, attempts have been madeto minimize the maltose interference effect by cloning new GlucDORenzymes with a greater specificity to glucose. However, progress has notresulted in a feasible solution.

In still yet another example, Abbott Laboratories' PCx/TrueMeasure™system utilizes amperometry coupled with a Nicotinamide AdenineDinucleotide (NAD)-dependent Glucose Dehydrogenase enzyme (GDH/NAD) toprovide a system substantially free from maltose interference. However,the amperometric system can provide inaccurate readings because of bloodoxygen level (pO2) interference, an effect that causes readings ofglucose levels to vary due to a variety of factors including where thesample is taken. For example, blood oxygen levels can vary substantiallydepending on whether the sample is capillary, venous, or arterial blood.Consequently, to minimize oxygen interference, a user of this systemmust indicate the source or nature of the blood sample prior tosampling. As can be appreciated, requiring the user to enter informationregarding the blood sample provides an additional source of error if theinformation is erroneously entered by the user.

In addition to slowing enzyme activity, vial abuse can also result in anincrease of background current, sometimes referred to as “blankcurrent”, when readings are taken. A variety of sources for backgroundor blank current exist. For instance, it is desirable that mediators,which are used to transfer electrons from the enzyme to the electrode,be in an oxidized state before the sensor is used. Over time the heatand/or humidity from the vial abuse will tend to reduce the mediator. Ifpart of the mediator is in a reduced form before the sensor is used, aportion of the current will result from the working electrode oxidizingthe reduced form of the mediator. The resulting background or blankcurrent will tend to bias the mediator, which in turn can lead toinaccurate results. Other impurities in the reagent can also increasebackground or blank current problems.

Amperometric sensors have been proposed that use a “burn-off” approachto address the blank current problem. In this approach, two DC signalsare applied to the sensor. The first DC signal, or burn-off signal, isused to consume or oxidize any species responsible for the blank currentin the same diffusion layer that is later used to analyze the analyte.Afterwards, the second signal, or analysis signal, is used to analyzethe analyte levels. Both the burn-off and analysis potentials have thesame polarity. Although this burn-off technique reduces the effect ofblank or background current, it does so at the expense of partiallyoxidizing (or reducing) the analyte to be measured, thereby reducing thenoise-to-signal ratio of the sensor. Moreover, such techniques havefailed to compensate for variations in reaction time caused by factorslike temperature and enzymes with slow/variable reaction velocities, toname a few examples. In addition, the enzymes used in such sensors tendto be susceptible to maltose interference.

In view of the above, it is desirable for a biosensor utilized tomeasure an analyte (such as glucose) to have a chemistry matrix withincreased specificity for the analyte, and to be capable of minimizinginterferences resulting from fluctuations in oxygen levels and frominterfering substances such as maltose. It is further desirable toprovide a chemistry matrix that is photochemically stable.

BRIEF SUMMARY

Embodiments of the compositions, methods and devices disclosed hereininvolve a chemistry matrix having increased specificity for an analyte(such as glucose) that is capable of minimizing interferences resultingfrom fluctuations in oxygen levels and from related substances (such asmaltose). Broadly, the chemistry matrix comprises glucose dehydrogenase,nicotinamide adenine dinucleotide, a phenazine derivative and anitrosoaniline. Preferred embodiments of the chemistry matrix aretypically stable at a pH of from about 6.5 to about 8.5 and arephotochemically stable. A chemistry matrix or components thereof areconsidered photochemically stable if the matrix or a component thereofremains colorless upon exposure to ordinary fluorescent lighting for atleast one hour.

A first aspect of the present disclosure provides for a chemistry matrixcomposition for use in analyzing the concentration of an analyte. Thischemistry matrix which is stable at a pH of from about 6.5 to about 8.5,is photochemically stable, and includes glucose dehydrogenase,nicotinamide adenine dinucleotide, an alkylphenazine quaternary salt,and a nitrosoaniline.

Suitable alkylphenazine quaternary salts useful in the presentdisclosure include, but are not limited to, 5-alkyl phenazinesillustrated by formula Ia:

-   -   where:    -   Z is selected from the group consisting of —(CH₂)_(m)COOH,        —NHAc, and —OY,    -   where m is an integer ranging from 0 to about 6 and Y is        selected from the group consisting of

where n is an integer ranging from about 1 to about 4, Q is —OH, —OR¹,—NH₂, —NHR², —NR²R³, —NH(CH₂)_(o)NR²R³, —NH(CH₂)_(o)OH, —NHCH₂CH₂—(OCH₂CH₂)_(o)-G, —(CH₂)_(o)NR²R³, —OCH₂CH₂)_(o)NR²R³ and —(OCH₂CH₂)_(o)OH,where G is —COOH, NR²R³, or

R¹ is a C₁ to C₆ alkyl group, R² and R³ are the same or different andeach represents a H or a C₁ to C₆ alkyl group, o is an integer rangingfrom about 1 to about 6; and X is an anion selected from the groupconsisting of halide, sulfate, alkyl sulfate, phosphate, phosphite,carboxylate, CF₃COO⁻, CH₃OSO₂ ⁻, C₂H₅OSO₂ ⁻, and CH₃SO₃ ⁻.

A second aspect of the present disclosure provides for a chemistrymatrix composition that can be used to determine the concentration of ananalyte where the chemistry matrix includes glucose dehydrogenase,nicotinamide adenine dinucleotide, a nitrosoaniline and aphotochemically stable 5-alkyl-1-carboxyalkoxyphenazine useful fordetermining the concentration of an analyte. The “carboxy group” in acarboxyalkoxyphenazine group includes carboxylic acids, their salts,esters, amides, nitriles, and other known derivatives of carboxylicacids. Particularly suitable 5-alkyl-1-carboxyalkoxyphenazines arerepresented by the formula IIa below.

where R⁶ is selected from the group consisting of —OH, —OR¹, —NH₂,—NHR², —NR²R³, —NH(CH₂)_(m)NR₂, —NH(CH₂)_(m) OH, —O(CH₂)_(m)NH₂, and—O(CH₂)_(m)OH, R¹ is a C₁ to C₆ alkyl group, R² and R³ are the same ordifferent and each represents a H or a C₁ to C₆ alkyl group, X⁻ is ananion selected from the group consisting of halide, sulfate, alkylsulfate, phosphate, phosphite, carboxylate, CF₃COO⁻, CH₃OSO₂ ⁻, C₂H₅OSO₂⁻, and CH₃SO₃ ⁻, and n and m are integers ranging from about 1 to about6.

A third aspect of the present disclosure provides for a furtherchemistry matrix composition for use in analyzing the concentration ofan analyte. This chemistry matrix includes:

(a) glucose dehydrogenase;

(b) nicotinamide adenine dinucleotide;

(c) a phenazine ethosulfate having the formula:

where R⁶ is selected from the group consisting of —OH, —OR¹, —NH₂,—NR²R³, —NH(CH₂)_(m)NH₂, —NH(CH₂)_(m) OH, —O(CH₂)_(m)NH₂, and—O(CH₂)_(m)OH, R² and R³ are the same or different and each represents aH or a C₁ to C₆ alkyl group, X⁻ is an anion selected from the groupconsisting of halide, sulfate, alkyl sulfate, phosphate, phosphite,carboxylate, CF₃COO⁻, CH₃OSO₂ ⁻, C₂H₅OSO₂ ⁻, and CH₃SO₃ ⁻, n is aninteger ranging from about 1 to about 6, m is an integer ranging fromabout 1 to about 6; and

(d) a nitrosoaniline derivative having the formula:

In this chemistry matrix, a phenazine of formula Ia can also besubstituted for the phenazine of formula II above.

A forth aspect of the present disclosure provides for a chemistry matrixcomposition useful for determining the concentration of an analyte. Thecomposition includes glucose dehydrogenase, nicotinamide adeninedinucleotide, a nitrosoaniline and a photochemically stable1-carboxyalkyloxy-5-alkylphenazine.

A fifth aspect of the present disclosure provides for a method for theelectrochemical analysis of an analyte in a liquid sample that involvesreacting the analyte with the matrix which includes glucosedehydrogenase, nicotinamide adenine dinucleotide, a nitrosoaniline and aphotochemically stable 1-carboxyalkyloxy-5-alkylphenazine to provide anelectro-active agent capable of producing an electrochemical response,providing a signal to produce an electrochemical response, measuring theelectrochemical response produced, and determining the analyteconcentration in the liquid sample based on the electrochemical responsemeasured. This method typically involves applying the sample to anapplication area of a biosensor having a test strip therein including anembodiment of the chemistry matrix described above, and quantifying theanalyte by reaction with the chemistry matrix. The method isparticularly effective in determining blood glucose levels ranging fromabout 20 mg/dL to about 600 mg/dL and can provide test times of about 5seconds or less. Test time refers to the time between applying a testsample to a test strip (or actually detecting the applying of the testsample) and when a test result is obtained and displayed on the displayof a monitoring device configured for use with the biosensor.

A sixth aspect of the present disclosure provides a method for theelectrochemical analysis of an analyte in a liquid sample, whichcomprises applying the sample to an application area of a biosensorhaving a test strip including the chemistry matrix, quantifying theanalyte by reaction with the chemistry matrix and displaying a result ofsaid quantifying on a display of a monitoring device configured for usewith said biosensor, wherein said chemistry matrix includes glucosedehydrogenase, nicotinamide adenine dinucleotide, a nitrosoaniline, anda 5-alkylphenazine quaternary salt having the formula:

where:

-   -   Z is selected from the group consisting of —(CH₂)_(m)COOH,        —NHAc, and —OY, where m can any integer ranging from 0 to about        6 and Y is selected from the group consisting of

where n is an integer ranging from about 1 to about 4, Q is —OH, —OR¹,—NH₂, —NHR², —NR²R³, —NH(CH₂)_(o)NR²R³, —NH(CH₂)_(o)OH, —NHCH₂CH₂—(OCH₂CH₂)_(o)-G, —(CH₂)_(o)NR²R³, —OCH₂CH₂)_(o)NR²R³ and —(OCH₂CH₂)_(o)OH,where G is —COOH, NR²R³, or

R¹ is a C₁ to C₆ alkyl group, R² and R³ are the same or different andeach represents a H or a C₁ to C₆ alkyl group, o is an integer rangingfrom about 1 to about 6; and X is an anion selected from the groupconsisting of halide, sulfate, alkyl sulfate, phosphate, phosphite,carboxylate, CF₃COO⁻, CH₃OSO₂ ⁻, C₂H₅OSO₂ ⁻, and CH₃SO₃ ⁻. Although avariety of nitrosoanilines can be utilized in the chemistry matrix forthis method, the nitrosoaniline illustrated by structure III isparticularly suitable.

Numerous advantages and additional aspects of the present disclosurewill be apparent from the description of the preferred embodiments anddrawings that follow.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a first top view of a test strip used with embodiments of thepresent disclosure and includes a cover layer of the test strip removed.

FIG. 2 is a second top view of the FIG. 1 test strip with the coverlayer in place.

FIG. 3 is a plot illustrating the linear nature of the blood/glucoseresponse determined with the new chemistry matrix described in Example1.

FIG. 4 is a plot illustrating the lack of oxygen interference at twooxygen levels and at glucose levels ranging from about 0 to about 120mg/dL.

FIG. 5 is a plot illustrating the lack of oxygen interference at twooxygen levels and at glucose levels ranging from about 0 to about 700mg/dL.

FIG. 6 is a plot illustrating the interference of maltose using aconventional test strip and the absence of interference with a teststrip based on the new chemistry matrix.

DETAILED DESCRIPTION

For the purpose of promoting an understanding of the principles of thedisclosure, reference will now be made to the embodiments illustrated inthe drawings and specific language will be used to describe the same. Itwill nevertheless be understood that no limitation of the scope of thedisclosure is thereby intended. Such alterations and furthermodifications in the illustrated device and such further applications ofthe principles of the disclosure as illustrated therein as wouldnormally occur to one skilled in the art to which the disclosure relatesare contemplated as within the scope of the disclosure. In particular,although the disclosure is discussed in terms of a blood glucose meter,it is contemplated that the invention can be used with devices formeasuring other analytes and other sample types. Such alternativeembodiments require certain adaptations to the embodiments discussedherein that would be apparent to those skilled in the art.

The present disclosure generally concerns a chemistry matrix that isused to determine the concentration of an analyte (such as, for example,glucose) in the presence of related components (such as for example,maltose) and varying levels of unrelated components (such as forexample, oxygen). As previously noted, both the presence of maltose andvariations in oxygen levels can interfere with the determination ofglucose carried out with a variety of electrochemical biosensors. Whilethe chemistry matrix and accompanying methods will be described belowwith reference to analyzing blood glucose levels, it should berecognized that the chemistry matrix and methods disclosed can be usedto analyze other types of analytes as well.

Embodiments of the present disclosure contemplate a new chemistry matrixfor use in determining the concentration of an analyte in a biologicalfluid. Typically, embodiments of the new chemistry matrix compositioninclude a glucose dehydrogenase, nicotinamide adenine dinucleotide, aphenazine derivative, and/or a nitrosoaniline, or derivative thereof.The different embodiments of the chemistry matrix are used with anelectrochemical biosensor to determine the concentration of an analyteafter a reaction occurs within the biosensor. Embodiments of phenazinederivatives include photochemically stable phenazine derivatives, suchas 1-substituted-5-alkyl phenazine quaternary salts. Further embodimentsare also contemplated. A method for determining the concentration of ananalyte using embodiments of the chemistry matrix disclosed is anotheraspect of the disclosure. The method can determine, for example, bloodglucose levels ranging from about 20 mg/L to about 600 mg/L at testtimes of about five seconds or less. The chemistry matrix can functionwell at a pH of from about 6.5 to about 8.5.

The new chemistry matrix will now be fully described in detail.Components of the new chemistry matrix can include an enzyme, acofactor, a reagent, and a mediator. When employed, the enzyme can beglucose dehydrogenase, the cofactor can be nicotinamide adeninedinucleotide, the reagent can be a phenazine derivative, and themediator can be a nitrosoaniline. Embodiments of phenazine derivativesinclude 5-alkylphenazine quaternary salts. It will be understood thatnitrosoaniline is an example of an indirect electron mediator in thesense that a nitrosoaniline molecule is not itself directly involved inan electron mediating sequence typically employed in electrochemicalanalyte detection and measurement methods; rather, nitrosoanilineparticipates in the sequence by reacting with glucose and othercomponents of the chemistry matrix to produce once or twice removedelectroactive reaction product, and it is the electroactive reactionproduct that is directly involved in the typical mediating sequence, asmay be more fully described and explained in U.S. Pat. Nos. 5,122,244and 5,286,362 (referenced above). Notwithstanding the foregoing,nitrosoaniline and/or its derivatives may be referred to as a mediatorherein and in other references created and/or prepared by applicant withthe understanding that nitrosoaniline is a precursor to the mediatordirectly involved in the electrochemical mediator sequence.

Phenazine Derivatives

Although not required, suitable phenazine derivatives are typicallychosen such that the derivatives are stable in the presence of light andparticularly at a pH range of from about 7 to about 8.5. Suitablephenazine derivatives include 5-alkylphenazine quaternary salts, such as5-alkyl-1-substituted phenazine quaternary salts of the type illustratedby formulas Ia described above and IIa and IIb provided below:

where R⁶ is selected from the group consisting of —OH, —OR¹, —NH₂,—NHR², —NR²R³, —NH(CH₂)_(m)NR₂, —NH(CH₂)_(m) OH, —O(CH₂)_(m)NH₂, and—O(CH₂)_(m)OH, R² is a C₁ to C₆ alkyl group, R¹ is C₁ to C₆ alkyl, R²and R³ are the same or different and each represents a H or a C₁ to C₆alkyl group, X⁻ is an anion selected from the group consisting ofhalide, sulfate, alkyl sulfate, phosphate, phosphite, carboxylate,CF₃COO⁻, CH₃OSO₂ ⁻, C₂H₅OSO₂ ⁻, and CH₃SO₃ ⁻, and n and m are integersranging from about 1 to about 6.Nitrosoanilines

Nitrosoanilines have the general formula III

in which R¹⁰ denotes hydrogen, halogen, alkoxy or alkylthio, and R¹¹represents an alkyl or a hydroxyalky residue and R¹² represents ahydroxyalkyl residue or R¹¹ and R¹² are the same or different and eachrepresents a dialkylaminoalkyl residue, a hydroxyalkyl, ahydroxyalkoxyalkyl or alkoxyalkyl residue optionally substituted by OHin the alkyl moiety or a polyalkoxyalkyl residue optionally substitutedby a hydroxy residue in the alkyl moiety, or R¹¹ and R¹² form analkylene residue interrupted by sulphur or nitrogen in which nitrogen issubstituted by an alkyl, hydroxyalkyl, hydroxyalkoxyalkyl,alkoxyhydroxyalkyl, dioxanylyl-alkyl or polyalkoxyalkyl residue, each ofwhich is itself optionally substituted in the alkyl moiety by a hydroxyresidue, or if R¹⁰ is in the ortho position to NR¹⁰R¹¹, R¹¹ alsotogether with R¹⁰ represents an alkylene residue wherein R¹² thenrepresents a hydroxyalkyl residue or, if the alkylene residue contains 3carbon atoms, it also optionally represents an alkyl residue. In thisconnection halogen denotes fluorine, chlorine, bromine or iodine.Fluorine and chlorine are typical halogens for R¹⁰. Alkyl, alkoxy oralkylthio are residues with 1-6 carbon atoms, with those containing 1-3carbon atoms being particularly suitable. The foregoing definition foralkyl also applies to the alkyl moiety in hydroxyalkyl,dialkylaminoalkyl, hydroxyalkoxy-alkyl, alkoxyalkyl, polyalkoxyalkyl,alkoxy-hydroxyalkyl and dioxanylyl-alkyl residues.

A dioxanylyl-alkyl residue is a residue in which a dioxan ring system isbound to an alkyl residue. It is typically a 1,4-dioxan ring system,i.e.

A polyalkoxyalkyl residue is an -alkyl-(alkoxy)_(p)-alkoxy residue inwhich p equals 1-10. Typically p equals 1-4; and more typically, pequals 1-3.

An alkylene residue is a straight-chained or branched residue,preferably straight-chained and can be either saturated or unsaturated,such as a saturated hydrocarbon chain consisting of 2-5, preferably 2-4,C-atoms with two free binding sites.

Within the meaning of an alkylene residue of R¹¹ and R¹² which isinterrupted by sulphur or nitrogen, a thiomorpholine or piperazineresidue formed by the inclusion of the nitrogen atom is suitable. Thepiperazine residue is especially suitable.

Within the meaning of an alkylene residue formed from R¹¹ and R¹², theindoline or 1,2,3,4-tetrahydroquinoline residue formed by the inclusionof the aromatic ring of the general formula V is suitable.

As the salt of a nitrosoaniline derivative according to the presentdisclosure of the general formula III, those of strong acids, inparticular mineral acids such as hydrochloric acid, sulphuric acid,nitric acid and phosphoric acid are suitable. Hydrochlorides areespecially suitable.

The following new nitrosoaniline derivatives are especially suitablecomponents of the present chemistry matrix:

a) 2,2′-[(3-fluoro-4-nitrosophenyl)imino]bis-ethanol,

b) 2,2′-[(3-chloro-4-nitrosophenyl)imino]bis-ethanol,

c) 2,2′-[(3-methoxy-4-nitrosophenyl)imino]bis-ethanol,

d) 2,2′-[(3-methylmercapto-4-nitrosophenyl)imino]bis-ethanol,

e) 2-[(2-hydroxyethoxy)ethyl-(4-nitrosophenyl)amino]ethanol,

f) 2-[(2-methoxyethoxy)ethyl-(4-nitrosophenyl)amino]ethanol,

g) 1-[N-(2-hydroxyethyl)-(4-nitrosoanilino)]-3-methoxy-2-propanol,

h)1-[N-(2-hydroxyethyl)-(4-nitrosoanilino)]-3-(2-hydroxyethoxy)-2-propanol,

i) 1-methyl-4-(4-nitrosophenyl)-piperazine,

j) 4-(4-nitrosophenyl)-1-piperazino-ethanol,

k) 5-nitroso-1-indoline ethanol,

l) 1-methyl-6-nitroso-1,2,3,4-tetrahydroquinoline,

m) 6-nitroso-3,4-dihydro-1(2H)quinoline ethanol and their salts

n) 2-[(2-hydroxyethyl-4-nitrosophenyl)amino]ethanol.

Of these the compounds a), d), e), f), g) and h) as well as their saltsare particularly suitable. Compound e) or its salts, in particular thehydrochloride, is especially suitable.

Preparation of Phenazine Component

One of the components in the chemistry matrix includes a phenazinederivative. The phenazine derivative can be selected from a variety ofdifferent substituted phenazines, however, 5-alkyl, 1-substitutedphenazines are useful in this present disclosure, most preferably5-ethyl-1-substituted phenazines. A variety of different linking groupsat position 1 of 5-alkylated phenazine are suitable. Suitablesubstituted phenazine include structure Ia, provided below:

-   -   where:    -   Z is selected from the group consisting of —(CH₂)_(m)COOH,        —NHAc, and —OY,    -   where m is an integer ranging from 0 to about 6 and Y is        selected from the group consisting of

where n is an integer ranging from about 1 to about 4, Q is —OH, —OR¹,—NH₂, —NHR², —NR²R³, —NH(CH₂)_(o)NR²R³, —NH(CH₂)_(o)OH, —NHCH₂CH₂—(OCH₂CH₂)_(o)-G, —(CH₂)_(o)NR²R³, —OCH₂CH₂)_(o)NR²R³ and —(OCH₂CH₂)_(o)OH,where G is —COOH, NR²R³, or

R¹ is a C₁ to C₆ alkyl group, R² and R³ are the same or different andeach represents a H or a C₁ to C₆ alkyl group, o is an integer rangingfrom about 1 to about 6; and X is an anion selected from the groupconsisting of halide, sulfate, alkyl sulfate, phosphate, phosphite,carboxylate, CF₃COO⁻, CH₃OSO₂ ⁻, C₂H₅OSO₂ ⁻, and CH₃SO₃ ⁻.

An especially suitable phenazine derivative can be illustrated byformula IIa provided below:

where R⁶ is selected from the group consisting of —OH, —OR², —NH₂,—NHR², —NR²R³, —NH(CH₂)_(m)NR₂, —NH(CH₂)_(m) OH, —O(CH₂)_(m)NH₂, and—O(CH₂)_(m)OH, R² is a C₁ to C₆ alkyl group, R¹ is C₁ to C₆ alkyl, R²and R³ are the same or different and each represents a H or a C₁ to C₆alkyl group, X⁻ is an anion selected from the group consisting ofhalide, sulfate, alkyl sulfate, phosphate, phosphite, carboxylate,CF₃COO⁻, CH₃OSO₂ ⁻, C₂H₅OSO₂ ⁻, and CH₃SO₃ ⁻, and n and m are integersranging from about 1 to about 6.

Reaction schemes 1-9, which follow, illustrate some typical reactionpathways which can be used to prepare suitable phenazine derivatives.

Scheme 1 represents the synthesis of the 5-ethyl-1-alkoxy carboxylicacid phenazine derivative (5). The preparation of this compound isdescribed in the literature [see Eur. J. Biochem, 179, 293-298 (1989)].However, the synthetic procedure was extensively modified to provide thenecessary quantities of the substituted phenazine (5).

1-Hydroxyphenazine (1) was purchased from TCI America. This was reactedwith ethyl 4-bromobutyrate in the presence of potassium carbonate and18-crown 6 in acetone under reflux conditions to give phenazine ethylester (3). The reaction of phenazine ethyl ester (3) with diethylsulfateas described in the literature did not provide any N-alkylated product.The reaction process for N-ethylation of phenazine ethyl ester (3) wasinvestigated by adding a base, such as potassium carbonate. Thus,reaction of phenazine ethyl ester (3) with diethylsulfate in thepresence of potassium carbonate at 100° C. for 18 h provided the5-N-ethyl 1-substituted carboxylic acid ethyl ester phenazine derivative(4). The resulting 5-N-ethyl phenazine ethyl ester was hydrolyzed bydilute hydrochloric acid to achieve the 5-ethyl-1-carboxybutyl phenazinederivative (5).

Scheme 2 represents the synthesis of a 1-alkoxy-substituted phenazinederivative (9) having a hydrophilic linker which can affect thephenazine's solubility. The availability of hydrophilic substitutedphenazines allows for a greater range of matrix formulations. The5-N-ethyl phenazine 1-hydroxybutyric acid derivative (5) is reacted with1-Ethyl N-3,3-dimethylaminopropyl carbodiimide and N-hydroxysuccinimideto give corresponding N-hydroxysuccinimide. This activated ester isreacted with the commercially available amino d-PEG₄ methyl ester(Quanta Biodesign, USA) in the presence of a tertiary base, such astriethylamine. Deprotection of a methyl ester is described in theliterature (see Greene, T. and Wuts, P., “Protective Groups in Organicsynthesis”, 2^(nd) edition, Wiley Intersciences, 1991). The methyl estergroup can be deprotected in the presence of an acid or a base to givephenazine derivative (9) with a PEG linker. Hydrolysis of the methylester with dilute hydrochloric acid has proven particularly suitable inthis phenazine chemistry system.

Scheme 3 describes the synthesis of a dimerized phenazine derivative(11). A chemistry matrix including phenazine derivative (11) typicallyprovides improved sensitivity. Phenazine derivative (11) can be preparedby reacting the activated ester (6) with 2,2-(ethylenedioxy)bisethylamine (Aldrich Chemical Company, USA), in the presence of atertiary base such as triethylamine or diisopropylethylamine and in asolvent such as dimethylformamide or tetrahydrofuran at a temperatureranging from 0° C. to room temperature. Particularly suitablebase/solvent combinations include triethylamine/dimethylformamide.

Scheme 4 illustrates the synthesis of an amino terminated 1-substitutedN-ethyl phenazine. O-Alkylation of 1-hydroxyphenazine can be carried outusing a terminal amino protected alkylating agent, such as for example,a phthalimido or a t-BOC derivative. The resulting t-Boc protected aminecan be deprotected under acidic conditions, such as trifluoroaceticacid, whereas the resulting phthalimido protected amine can bedeprotected in the presence of hydrazine or methylamine. A particularlysuitable method includes reacting 1-hydroxyphenazine withN-(4-bromobutyl)phthalimide (Acros Chemicals, USA), in the presence of abase, such as potassium carbonate in a solvent such as acetone, DMF, orTHF under reflux conditions. Acetone has been found to be a particularlysuitable solvent for this reaction. The resulting alkylated product (13)is reacted with diethylsulfate in the presence of potassium carbonate togive N-ethyl phthalimido protected phenazine (14). The phthalimido groupis deprotected with methylamine in methanol at room temperature to giveamino terminated 1-substituted N-ethyl phenazine (15).

Scheme 5 describes the synthesis of a hydroxyl terminated 1-substitutedphenazine derivative. 1-Hydroxy phenazine (1) is reacted with a hydroxyprotected alkylating agent, such as for example,(3-bromopropoxy)-tert-butyldimethylsilane (16), (Aldrich chemicalcompany, USA) in the presence of potassium carbonate in acetone underreflux conditions to give the tert-butyldimethylsilane (TBDMS) protectedphenazine derivative (17). N-alkylation of (17) is performed withdiethylsulfate in the presence of potassium carbonate to give N-ethyl1-substituted protected hydroxyl terminated phenazine derivative (18).Deprotection of TBDMS group is performed in the presence oftetrabutylammonium fluoride (TBAF) in THF at room temperature to give1-hydroxybutyl N-ethyl phenazine (19).

Scheme 6 illustrates the synthesis of N-ethyl phenazine 1-hydroxybutylmethyl ether. The phenazine ether (21) is prepared by the alkylation ofthe 1-hydroxyphenazine (1) with 4-methoxybutylbromide (Aldrich ChemicalCompany, USA) to give phenazine derivative (21). N-ethyl phenazine (22)is prepared by reaction of compound (21) with diethyl sulfate in thepresence of potassium carbonate.

Reaction scheme 7 illustrates the synthesis of a 1-carboxy N-ethylphenazine (29a) and 8-methyl 1-carboxy N-ethyl phenazine (29b)derivative. A mixture of aniline (or ortho toluidine) and2-bromo-3-nitrobenzoic acid is reacted in the presence of CuCl, Cupowder and N-ethylmorpholine in butane-2,3-diol at 70 to 80° C. for 8 to24 hours. This reaction mixture is diluted with 0.1 M NH₄OH solution andfiltered through a bed of celite. The resulting solution is slowlypoured over into 2N HCl to give N-phenyl-3-nitro anthranilic acid (25a)or the methyl substituted derivative (25b). Intermediates (25a) or (25b)are reacted with sodium borohydride in 2N NaOH solution under refluxconditions to effect ring closure and provide the sodium salt of aphenazine, which, upon acidification, provides (26a) or (26b). The acidchloride of the phenazine 1-carboxy derivative (or 8-methyl1-carboxyphenazine) is prepared by the reaction with thionyl chlorideand the resulting acid chloride is converted to methyl ester by thereaction with methanol in HCl. The resulting phenazine methyl ester(27a) or (27b) is alkylated with diethyl sulfate and potassium carbonateto give corresponding N-ethyl phenazine derivatives (28a) or (28b).Finally, the methyl ester group of phenazines (28a) or (28b) ishydrolyzed with dilute hydrochloric acid to give (29a) or (29b).

Scheme 8 provides a synthetic route to a 1-acetamido-N-ethyl phenazinederivative (35). A 3-amino-catechol hydrochloride (30) is reacted withsilver oxide and anhydrous sodium sulfate in ethyl acetate to give3-amino-1,2-quinone (31), which is further reacted in situ with orthophenylenediamine (32) to give 1-amino phenazine (33). The aminophenazine (33) is acetylated with acetic anhydride in acetic acid togive 1-acetamido phenazine (34). The resulting 1-acetamido phenazine isreacted with diethyl sulfate in the presence of potassium carbonate togive N-ethyl-1-acetamido-phenazine (35).

Scheme 9 provides a synthetic route to the 1-carboxymethyl-5-N-ethylphenazine derivative (42). The bromo derivative (37) is formed byreacting 1-methyl phenazine (36) (available from Apin Chemicals, UK)with N-bromosuccinimide in the presence of benzoyl peroxide and asolvent. Carbon tetrachloride is a particularly suitable solvent for thebromination reaction. Reaction of the bromide (37) with KCN in asuitable solvent, such as, for example, DMF, gives the 1-cyanomethylphenazine derivative (38). Acid hydrolysis of the cyanophenazine (38)provides the 1-carboxymethyl phenazine (39). The acid group of compound(39) is converted to a methyl ester by reaction with methanol and HCl togive the phenazine ester (40). Ester (40) can be N-alkylated withdimethyl sulfate in the presence of potassium carbonate to give the5-ethyl phenazine ester (41). Hydrolysis of the ester (41) with dilutehydrochloric acid provides the 5-ethyl-1-carboxymethyl phenazine (42).

Preparation of Nitrosoaniline Component

The compounds of the general formula III can be produced by reacting acompound of the general formula VI, in which R⁴, R⁵ and R⁶ have the samemeaning as in compounds of the general formula III, with nitrite.Details concerning formula III are provided above.

An analogous process is known from J. J. D'Amico et al., J. Amer. Chem.Soc. 81, 5957 (1959).

Alkali nitrite is typically used as the nitrite, in which lithium,sodium, potassium, rubidium or cesium is possible as the alkali metal;sodium nitrite and potassium nitrite are particularly suitable. Sodiumnitrite is especially suitable. The reaction typically takes place in anacid medium at low temperature. It is advantageous when the temperatureis below 10° C., preferably between −10 and +5° C.

It is advantageous when the reaction of a compound of the generalformula VI with nitrite takes place in an aqueous medium. A suitable pHfor the medium is less than 3, and less than 2 is particularly suitable.

In a one embodiment for the reaction, a compound of the general formulaVI or a salt thereof, such as a salt of a mineral acid such ashydrochloric acid, sulphuric acid, nitric acid or phosphoric acid, isfirst added to an aqueous acidic medium and cooled.

Then, nitrite, typically in a dissolved form, is added while maintainingthe reaction mixture at a low temperature. It is advantageous when anaqueous medium is also used as the solvent for the nitrite. Afteraddition of the nitrite the reaction mixture is kept at a lowtemperature until the reaction is completed. In order to process thereaction mixture it is typically extracted with an organic solvent andthe product is isolated from the extract.

Chemistry Matrix

A first embodiment of the chemistry matrix includes glucosedehydrogenase, nicotinamide adenine dinucleotide, a 5-ethyl phenazinequaternary salt, and a nitrosoaniline. Although not required, suitablequaternary salts are typically photochemically stable. Examples ofphotochemically stable 5-ethyl quaternary salts are illustrated byformula Ia and IIb:

-   -   where:    -   Z is selected from the group consisting of —(CH₂)_(m)COOH,        —NHAc, and —OY,    -   where m is an integer ranging from 0 to about 6 and Y is        selected from the group consisting of

where n is an integer ranging from about 1 to about 4, Q is —OH, —OR¹,—NH₂, —NHR², —NR²R³, —NH(CH₂)_(o)NR²R³, —NH(CH₂)_(o)OH, —NHCH₂CH₂—(OCH₂CH₂)_(o)-G, —(CH₂)_(o)NR²R³, —OCH₂CH₂)_(o)NR²R³ and —(OCH₂CH₂)_(o)OH,where G is —COOH, NR²R³, or

R¹ is a C₁ to C₆ alkyl group, R² and R³ are the same or different andeach represents a H or a C₁ to C₆ alkyl group, o is an integer rangingfrom about 1 to about 6; and X is an anion selected from the groupconsisting of halide, sulfate, alkyl sulfate, phosphate, phosphite,carboxylate, CF₃COO⁻, CH₃OSO₂ ⁻, C₂H₅OSO₂ ⁻, and CH₃SO₃ ⁻; and

where R⁶ is selected from the group consisting of —OH, —OR², —NH₂,—NHR², —NR²R³, —NH(CH₂)_(m)NR₂, —NH(CH₂)_(m) OH, —O(CH₂)_(m)NH₂, and—O(CH₂)_(m)OH, R² is a C₁ to C₆ alkyl group, R² and R³ are the same ordifferent and each represents a H or a C₁ to C₆ alkyl group, X⁻ is ananion selected from the group consisting of halide, sulfate, alkylsulfate, phosphate, phosphite, carboxylate, CF₃COO⁻, CH₃OSO₂ ⁻, C₂H₅OSO₂⁻, and CH₃SO₃ ⁻, and n and m are integers ranging from about 1 to about6.

A further embodiment of the chemistry matrix includes a phenazine alkylquaternary salt having the formula illustrated in formulas IIa or IIb,provided above. Suitable quaternary salts are typically photochemicallystable and stable at a pH of from about 6.5 to about 8.5. Additionalcomponents of this embodiment can include glucose dehydrogenase,nicotinamide adenine dinucleotide and/or a nitrosoaniline. Suitablenitrosoanilines are represented by formula III, provided above.

A still further embodiment of the chemistry matrix includes glucosedehydrogenase, nicotinamide adenine dinucleotide, a 5-ethyl phenazinequaternary salt represented by formula IIb provided above, and a1,4-nitrosoaniline represented by formula VII.

Suitable phenazine quaternary salts are typically photochemicallystable.

A still further embodiment of the chemistry matrix composition includesa photochemically stable 5-alkyl phenazine quaternary salt. Thecomposition can additionally contain glucose dehydrogenase, nicotinamideadenine dinucleotide, and a nitrosoaniline. Suitable phenazinequaternary salts are represented by formula I, provided above. Suitablenitrosoanilines are represented by formula III, provided above.

Another embodiment includes a method or test for determining theconcentration of an analyte sample (particularly, glucose in blood)utilizing embodiments of the chemistry matrix. The method includes usinga biosensor and test strip configuration as previously described. Thechemistry matrix is used in a chemical reaction to determine theconcentration of the analyte. Certain methods utilize a chemistry matrixthat includes glucose dehydrogenase, nicotinamide adenine dinucleotide,a 5-alkyl phenazine quaternary salt, and a nitrosoaniline. Certain othermethods utilize a 5-alkylphenazine quaternary salt represented byformula IIb and yet other methods utilize a nitrosoaniline representedby formula VII. The method described herein can be used to analyzesamples containing from about 20 mg/L to about 600 mg/L of blood glucosein a test time of about 5 seconds or less. Optimum results can beobtained upon utilizing the chemistry matrix within a pH range of fromabout 6.5 to about 8.5.

Reference to the drawings further discloses aspects of the presentdisclosure. An amperometric electrochemical analyte sensor 20, suitablefor use with the chemistry and methods described herein, is illustratedin FIGS. 1 and 2. It should be recognized that the sensor 20 in FIGS. 1and 2 is merely an example of a type of sensor that can be used inconjunction with the chemistry and method according to the presentdisclosure, and that other types of sensors with differentconfigurations can similarly be used. For example, although the sensorillustrated in FIGS. 1 and 2 has electrodes formed in an interdigitatingarray, sensors having a different configuration or with additionalelectrodes can be used with the disclosed chemistry matrix and method.As another example, the electrodes in the illustrated embodiment have aco-planar configuration, but it should be appreciated that theelectrodes 22 in other embodiments can have other configurations, suchas a facing construction. For the sake of brevity as well as clarity,not all of the features of the sensor system will be described in detailbelow, but reference is made to examples of other types of sensors withwhich the inventive chemistries and methods are useful, including thosedescribed in U.S. Pat. Nos. 5,989,917; 6,270,637; and U.S. PublishedApplication No. 2003/0155237 A1, all of which are hereby incorporated byreference in their entireties.

Turning now to FIG. 1, the sensor 20 includes an interdigitated array ofelectrodes 22 disposed on flexible substrate 24. One of the electrodes22 in the pair acts as a working electrode, and the other electrode actsas a counter electrode. However, as indicated before, the electrodes 22in accordance with one embodiment of the present disclosure can switchroles. That is, an electrode 22 at one time may act as a workingelectrode and may at another time act as a counter electrode. In theillustrated embodiment, two electrodes 22 are shown, but it should berecognized that the sensor 20 in other embodiments can include moreelectrodes. The electrodes 22 are connected to electrically-conductiveconnectors 26 that include contact pads 28 located on the surface of theflexible substrate 24, where the contact pads 28 are available to becontacted to an external electronic circuit, such as a meter. Theconnectors 26 also include connector portions 30, which connectelectrode elements at the array 22 to the pads 28 and which maytypically be covered by an insulating layer.

Referring to FIG. 2, non-conductive spacer layer 32 is disposed over thesubstrate 24 and connector portions 30 of the connectors 26. The spacerlayer 32 defines a capillary sample chamber 34, and the sample chamber34 has an inlet opening in which the fluid sample is drawn into thesample chamber 34. A reagent layer 35 is disposed over the array 22within the sample chamber 34. The reagent layer 35 will be described infurther detail below but is configured to analyze the fluid sample. Foil36 covers the spacer 32 and a portion of capillary chamber 34 except foran air vent 38, which is used to vent air from the chamber 34.

Preparation of Matrix Solutions

Standard Matrix Solution

A stock buffer solution was prepared by adding 25.148 g of Pipessesquisodium salt, 0.125 g of Triton X-100, and 2.40 g of Trehalose to400 mL of double distilled water and adjusting the solution's pH to7.00. This solution was added to a 500 mL volumetric flask and dilutedwith double distilled water to make 500 mL of solution. Preparation ofthe buffer/polymer solution was completed by combining 396 grams of theinitial buffer solution with 2 g of polyethylene oxide (300K) and 2 g ofNatrosol 250M. Upon standing overnight all solids had dissolved and thesolution was ready for use.

A matrix solution was prepared from the stock buffer solution by: (a)adding the following ingredients to a 25 mL speed mixing cup containing11.198 g of the buffer stock solution in a serial fashion and speedmixing for 1 minute at 33,000 rpm after each addition: 0.5592 g of KCl,0.1824 g of NAD grade 1 and 0.0913 g of the substituted nitrosoaniline(Structure VII); (b) adjusting the pH to 7.00; and (c) adding 0.0163 gof 1-(3-carboxypropyloxy)-5-ethylphenazine (5 from Scheme 1) tocontainer mixing for 1 minute at 33,000 rpm and finally, add 0.6574 g ofglucose dehydrogenase enzyme and speed mixing for 2 minute at 33,000rpm.

Additional Matrix Solutions Utilizing Alternative Phenazines

Matrix solutions can similarly be prepared utilizing the method providedabove by substituting alternative 1-substituted phenazine derivativesfor 1-(3-carboxypropyloxy)-5-ethyl phenazine or by similarlysubstituting alternative nitrosoanilines for the nitrosoanilinerepresented by formula VI. Suitable phenazine derivatives are typicallyphotochemically stable. Although 1-ethyl-5-substituted phenazinequaternary salts illustrated by formula II can be more suitable, other1-alkyl-5-substituted phenazine quaternary salts illustrated by formulaI, can also be utilized.

Additional Matrix Solutions Utilizing Alternative Nitrosoanilines

Matrix solutions can also be prepared by the method provided above bysubstituting nitrosoanilines represented by formula III for thenitrosoaniline represented by formula VII.

Preparation of Test Strips

Cards of ACCU-CHEK® Aviva brand electrodes with spacer and capillarydesign were charged with about 1.8 μL of the basic matrix solutiondescribed above in each electrode channel and dried at about 45° C. forabout 1 minute. The dried cards were stored in a dry atmosphereovernight and strips of hydrophilic top foils were manually laminatedonto the spacer layer. The cards were cut into appropriate strips andstored in desiccated vials until used. This method can similarly be usedto prepare test strips based on the several matrix solutions describedabove.

Examination of Whole Blood Dose Responses

Whole blood samples containing seven different levels of glucose (about50 to about 600 mg/dL) were measured utilizing the test strips preparedabove from the basic matrix solution utilizing about a 2.5 second delayand about a 2.5 second read, meaning that a signal was applied about 2.5seconds after contacting the sample with the test strip and a readingwas taken about 2.5 seconds after application of the signal. The meancurrent was measured at about 5 seconds after contacting the sample withthe test strip to provide a linear relationship between current andglucose concentration. The establishment of a linear relationshipbetween glucose concentration and current read facilitates utilizationof the chemistry matrix to analyze an analyte such as glucose. Theresults are provided in FIG. 3. Similar results are obtained utilizingtest strips prepared from the several matrix solutions described above.

Matrix Performance at Different Oxygen Levels

Blood glucose samples having glucose levels ranging from about 0 toabout 110 mg/dL were saturated with oxygen at about 39 mm Hg and atabout 100 mm Hg of oxygen and the glucose levels determined using thetest strips described above prepared from the basic matrix solution. Thesamples were run with about a 3 second delay and about a 2 second read.The mean current at about 5 seconds was determined and plotted. Neitheroxygen level affected the glucose measurement as illustrated in the plotprovided in FIG. 4. Similar results are obtained upon repeating thisexample with test strips prepared from the alternative matrix solutionsdescribed above.

In a subsequent test, blood samples having glucose levels ranging fromabout 50 to about 600 mg/L were saturated with oxygen at about 42 mm Hgand about 135 mm Hg of oxygen and the glucose levels measured using thetest strips prepared above from the basic matrix solution. Neitheroxygen level affected the glucose measurement over this widerconcentration range as illustrated in FIG. 5. Similar results areobtained upon repeating this example with test strips prepared from thealternative matrix solutions described above.

Maltose Interference Studies

A maltose stock solution was prepared by adding 21 mM of maltose to 200mL of saline solution. Six blood samples containing glucose levelsranging from about 10 to about 550 mg/L were prepared. Each sample wassplit into two portions and to one series of samples was added 0.05 mLof a maltose stock solution for each 1 mL portions of test sample. Tothe second series of samples, an equal volume of a saline solution wasadded to 1 mL portions of the test sample. The glucose content of thesamples was determined with a standard ACCU-CHEK® Aviva brand test stripand a test strip having generally similar structural elements, butcontaining the new chemistry matrix described above. As illustrated inFIG. 6, maltose inflated the measurements made with the standardACCU-CHEK® Aviva brand strip, whereas the determinations made with thenew chemistry matrix were substantially the same as samples that did notcontain maltose. Similar results are obtained upon repeating this studywith test strips prepared from the alternative matrix solutionsdescribed above. ACCU-CHEK® and Aviva are registered U.S. trademarks ofRoche Diagnostics Gmbh CORPORATION FED REP GERMANY, Sandhofer Strasse,116 Mannheim FED REP GERMANY D-68305.

While the disclosure has been illustrated and described in detail in thefigures and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly selected embodiments have been shown and described and that allchanges, modifications and equivalents that come within the spirit ofthe disclosures described heretofore and/or defined by the followingclaims are desired to be protected. In addition, all publications citedherein are indicative of the level of skill in the art and are herebyincorporated by reference in their entirety as if each had beenindividually incorporated by reference and fully set forth.

What is claimed is:
 1. A method for determining the concentration of ananalyte in a liquid sample with reduced interference from otherconstituents present with the analyte, which comprises reacting theanalyte with a matrix comprising glucose dehydrogenase, nicotinamideadenine dinucleotide, a nitrosoaniline and a 5-alkylphenazine quaternarysalt to provide an electro-active agent, wherein said matrix isphotochemically stable, measuring an electrochemical response producedby the electro-active agent, and determining the analyte concentrationin the liquid sample based on the electrochemical response measured. 2.The method of claim 1, wherein the analyte is glucose and the samplecontains from about 20 mg/dL to about 600 mg/dL of blood glucose.
 3. Themethod of claim 1 which comprises applying the sample to an applicationarea of a biosensor having a test strip including the matrix anddisplaying the concentration on a display of a monitoring deviceconfigured for use with said biosensor, wherein said 5 alkylphenazinequaternary salt has the formula:

where Z is selected from the group consisting of —(CH₂)_(m)COOH, —NHAc,and —OY, where m is an integer ranging from 0 to about 6 and Y isselected from the group consisting of

where n is an integer ranging from about 1 to about 4; Q is —OH, —OR¹,—NH₂, —NHR², —NR²R³, —NH(CH₂)_(o)NR²R³, —NH(CH₂)_(o)OH,—NHCH₂CH₂—(OCH₂CH₂)_(o)-G, —(CH₂)_(o)NR²R³, (—OCH₂CH₂)_(o)NR²R³and—(OCH₂CH₂)_(o)OH; and G is —COOH, NR²R³, or

R¹ is a C₁ to C₆ alkyl group, R² and R³ are the same or different andeach represents a H or a C₁ to C₆ alkyl group, o is an integer rangingfrom about 1 to about 6; and X is an anion selected from the groupconsisting of halide, sulfate, alkyl sulfate, phosphate, phosphite,carboxylate, CF₃COO⁻, CH₃OSO₂ ⁻, C₂H₅OSO₂ ⁻, and CH₃SO₃ ⁻.
 4. The methodof claim 3, wherein said determining can be carried out for said samplehaving a blood glucose level ranging from about 20 to about 600 mg/dL.5. The method of claim 3, wherein said displaying occurs within about 5seconds or less after applying said sample.
 6. The method of claim 1wherein said 5-alkylphenazine quaternary salt has the formula:

where Z is selected from the group consisting of —(CH₂)_(m)COOH, —NHAc,and —OY, where m is an integer ranging from 0 to about 6 and Y isselected from the group consisting of

where n is an integer ranging from about 1 to about 4; Q is —OH, —OR¹,—NH₂, —NHR², —NR²R³, —NH(CH₂)_(o)NR²R³, —NH(CH₂)_(o)OH,—NHCH₂CH₂—(OCH₂CH₂)_(o)-G, —(CH₂)_(o)NR²R³, (—OCH₂CH₂)_(o)NR²R³ and—(OCH₂CH₂)_(o)OH; and G is —COOH, NR²R³, or

R¹ is a C₁ to C₆ alkyl group, R² and R³ are the same or different andeach represents a H or a C₁ to C₆ alkyl group, o is an integer rangingfrom about 1 to about 6; and X is an anion selected from the groupconsisting of halide, sulfate, alkyl sulfate, phosphate, phosphite,carboxylate, CF₃COO^(—), CH₃OSO₂ ^(—), C₂H₅OSO₂ ^(—), and CH₃SO₃ ^(—).7. The method of claim 6, wherein R¹ is C₂H₅.
 8. The method of claim 7,wherein said nitrosoaniline has the formula:


9. The method of claim 8, wherein Z is —OY; Y is —(CH₂)_(n)COOQ; Q isOH; and n=3.
 10. The method of claim 8, wherein Z is —OY; Y is—(CH₂)_(n)COOQ; Q is —NHCH₂CH₂—(OCH₂CH₂)_(o)-G; G is —COOH; and o is 4.11. The method of claim 8, wherein Z is —OY; Y is —(CH₂)_(n)Q; Q is —NH₂and n is
 4. 12. The method of claim 8, wherein Z is —OY; Y is —(CH₂)_(n)Q; Q is —OH and n is
 3. 13. The method of claim 8, wherein Z is —OY; Yis —(CH₂)_(n) Q; Q is —OR²; R² is Me; and n is
 4. 14. The method ofclaim 8, wherein Z is —(CH₂)_(m)COOH and m is
 1. 15. The method of claim8, wherein Z is —NHAc.
 16. The method of claim 6 in which the5-alkylphenazine quaternary salt is a 5-ethylphenazine salt.
 17. Themethod of claim 16 in which the 5-ethylphenazine salt is1-(3-carboxypropyloxy)-5-ethylphenazine.
 18. The method of claim 1,wherein the nitrosoaniline is a compound having the formula:

and, R¹⁰ denotes hydrogen, halogen, alkoxy or alkylthio, R¹¹ representsan alkyl or a hydroxyalky residue and R¹² represents a hydroxyalkylresidue, R¹¹ and R¹² are the same or different and each represents adialkylaminoalkyl residue, a hydroxyalkyl, a hydroxyalkoxyalkyl oralkoxyalkyl residue, or R¹¹ and R¹² form an alkylene residue interruptedby sulphur, nitrogen, or a substituted nitrogen residue, or provided ifR¹⁰ is in the ortho position to NR¹⁰R¹¹,R¹¹ also together with R¹⁰represents an alkylene residue wherein R¹² then represents ahydroxyalkyl residue or, R¹⁰ and R¹² are the same or different and eachrepresents a hydroxyalkyl residue or a salt thereof.
 19. The method ofclaim 18 wherein said nitrosoaniline has the formula:


20. The method of claim 1 in which the 5-alkylphenazine quaternary salthas the formula:

where R⁶ is selected from the group consisting of —OH, —OR¹, —NH₂,—NHR², —NR²R³, —NH(CH₂)_(m)NR₂, —NH(CH₂)_(m)OH, —O(CH₂)_(m)NH₂, and—O(CH₂)_(m)OH, R¹ is a C₁ to C₆ alkyl group, R² and R³ are the same ordifferent and each represents a H or a C₁ to C₆ alkyl group, X⁻is ananion selected from the group consisting of halide, sulfate, alkylsulfate, phosphate, phosphite, carboxylate, CF₃COO⁻, CH₃OSO₂ ⁻, C₂H₅OSO₂⁻, and CH₃SO₃ ⁻, and n and m are integers ranging from about 1 to about6.
 21. The method of claim 20, wherein R¹ is C₂H₅.
 22. The method ofclaim 21, wherein said nitrosoaniline has the formula:

and, R¹⁰ denotes hydrogen, halogen, alkoxy or alkylthio, R¹¹ representsan alkyl or a hydroxyalkyl residue, and R¹² represents a hydroxyalkylresidue, R¹¹ and R¹² are the same or different and each represents adialkylaminoalkyl residue, a hydroxyalkyl, a hydroxyalkoxyalkyl oralkoxyalkyl residue, or R¹¹ and R¹² form an alkylene residue interruptedby sulphur, nitrogen, or a substituted nitrogen residue, or provided ifR¹⁰ is in the ortho position to NR¹⁰R¹¹, R¹¹ also together with R¹⁰represents an alkylene residue wherein R¹² then represents ahydroxyalkyl residue or, R¹⁰ and R¹² are the same or different and eachrepresents a hydroxyalkyl residue or a salt thereof.
 23. The method ofclaim 22, wherein said nitrosoaniline has the formula:


24. A method for determining the concentration of an analyte in a liquidsample with reduced interference from other constituents present withthe analyte, which comprises reacting the analyte with a matrixcomprising glucose dehydrogenase, nicotinamide adenine dinucleotide, anitrosoaniline and a photochemically stable1-carboxyalkyloxy-5-alklyphenazine to provide an electro-active agent,wherein said matrix is photochemically stable, measuring anelectrochemical response produced by the electro-active agent, anddetermining the analyte concentration in the liquid sample based on theelectrochemical response measured.
 25. The method of claim 24, whereinthe nitrosoaniline has the formula:

wherein, R¹⁰ denotes hydrogen, halogen, alkoxy or alkylthio, R¹¹represents an alkyl or a hydroxyalky residue, and R¹² represents ahydroxyalkyl residue, R¹¹ and R¹² are the same or different and eachrepresents a dialkylaminoalkyl residue, a hydroxyalkyl, ahydroxyalkoxyalkyl or alkoxyalkyl residue, or R¹¹ and R¹² form analkylene residue interrupted by sulphur, nitrogen, or a substitutednitrogen residue, or provided if R¹⁰ is in the ortho position toNR¹¹R¹², R¹¹ also together with R¹⁰ represents an alkylene residuewherein R¹² then represents a hydroxyalkyl residue.
 26. The method ofclaim 25, wherein the 5-alkyl-1-carboxyalkyloxyphenazine is a compoundhaving the formula:

wherein where R⁶ is selected from the group consisting of —OH, —OR¹,—NH₂, —NHR², —NR²R³, —NH(CH₂)_(m)NR₂, —NH(CH₂)_(m)OH, —O(CH₂)_(m)NH₂,and —O(CH₂)_(m)OH, R² is a C₁ to C₆ alkyl group, R¹ is C₁ to C₆ alkyl,R² and R³ are the same or different and each represents a H or a C₁ toC₆ alkyl group, X⁻ is an anion selected from the group consisting ofhalide, sulfate, alkyl sulfate, phosphate, phosphite, carboxylate,CF₃COO³¹ , CH₃OSO₂ ⁻, C₂H₅OSO₂ ⁻, and CH₃SO₃ ⁻, and n and m are integersranging from about 1 to about 6.